Field
The present disclosure relates to methods and kits for in vitro testing of irritants.
Description of the Related Art
Traditionally, ocular irritation testing has been conducted using rabbits. A test substance is applied to the conjunctival sack of rabbits under restraint for 4 hours, followed by a 14 or 21-day test period. After the first hour and on days 1, 2, 3, 4, 7 and 14 or 21, the immune response—i.e., redness, swelling and discharge, as well as tissue damage, are evaluated in three areas of the eye: the cornea, iris and conjunctiva. One purpose of this test is to evaluate any opacity caused by the test substance in the cornea of the eye and to measure it. Any disruption to the pigmented iris surrounding the pupil, which is deeper within the eye than the cornea, is also measured, and the conjunctiva or tissue surrounding the eye is evaluated for redness, chemosis and discharge.
The extent of damage, at each organ location are evaluated and scored. These scores are used to classify a test substance as either non-irritant, irritant or corrosive. Ocular irritation is distinguished from ocular corrosion based on reversibility. If, after 14 or 21 days, there is still a response, the substance is classified as corrosive but if the effect has reversed, the substance is classified as an irritant. A non-irritant induces little measurable change at each tissue site, whereas an irritant induces swelling and lesions at one or more sites. In addition, diffuse redness and distinct blood vessel damage are evaluated at the conjunctiva. At the cornea, opacity is evaluated. Corrosives induce similar types of damage, but the damage will never fully reverse which may cause scarring or loss of vision.
There are two predominant modern classification systems. The Environmental Protection Agency (EPA) protection system is used predominantly within the United States, while the Globally Harmonized System (GHS) is used predominantly in international commerce. The EPA and GHS classify irritants based on the severity of irritation as depicted Table 1 below.
TABLE 1SevereClassificationMildModerateIrritationSystemNo IrritationIrritationIrritation(Corrosion)EPACategory IVCategory IIICategory IICategory IGHSNCCategory 2Category 2Category 1(NotClassified)
Irritation testing is also used to satisfy U.S. FDA and international safety labeling requirements and plays an important role in commercial product liability and consumer product satisfaction. Guidance documents produced by the Organization for Economic Trade and Development (OECD) are available to coordinate international trade. The OECD describes the standard rabbit eye test, which is the primary method for eye safety evaluation, and Safety Data Sheet documentation which accompanies hazardous chemicals and products.
The restraint of the animals as well as the test duration required by traditional methods of ocular irritation testing has raised public concern over unnecessary animal suffering. There is broad-based objection and aggressive lobbying against cosmetic and personal care product testing using animals. The “not tested on animals” label appeals to a large segment of consumers. In addition, US regulators discourage animal testing for ocular irritation, and Europe has banned animal testing for most consumer product labeling. Interestingly, at the same time that animal testing was banned in 2013, Europe also will mandate safety guidance labeling; the United States is expected to follow suit.
There are a limited number of ocular irritation tests that do not require the use of animals. These tests include cell culture-based tests, tests based on excised animal eyes, slugs, egg-based tests, and one other existing biochemical tests that measures a single water soluble variable.
Cell culture-based tests typically involve applying the test substance to a differentiated human tissue grown in a petri dish, to determine its toxicity based on the degree of cells killed by the substance after a fixed time. Tests based on fertilized eggs, or the Hen's Egg Test Chorioallantoic Membrane (HET-CAM), measure changes to the vessels that extend from the developing yolk to the air cell within the egg; this primitive respiratory tissue or chorioallantoic membrane is the CAM. These vessels, present early in development before the egg is considered an animal, can be used to determine the irritancy and corrosivity of a test substance. It has been suggested that the vessels in the CAM are similar to those in the conjunctiva. When the test is conducted, the substance is applied to the CAM and changes to the vessels—typically lysis, coagulation and hemorrhage, are measured and used to predict ocular irritancy and corrosivity.
There remains an unmet need for an accurate cell free test for the assessment of ocular irritation.